WASP and SCAR Are Evolutionarily Conserved in Actin-Filled Pseudopod-Based Motility

Published May 4, 2017 JCB: Article WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility Lillian K. Fritz-Laylin, Samuel J. Lord, and R. Dyche Mullins Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143 Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call “α-motility.” Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE—activators of branched actin assembly—make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP’s role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with Downloaded from actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin. Introduction on June 21, 2017 Eukaryotic cells move using several distinct modes of locomo- leading-edge membrane, but in these cells, the force-generating tion, including crawling and fagella-driven swimming. The ste- polymer networks are composed of major sperm protein rather reotyped architecture of fagella and the conservation of their than actin (Rodriguez et al., 2005). Given this variety of crawling protein components make the evolutionary conservation of cell behaviors, it is clear that one cannot simply assume that the under- swimming clear. In contrast, “crawling motility” is a collection of lying molecular mechanisms are the same. distinct processes whose evolutionary relationships are not well The best-understood mode of crawling is the slow (1–10 understood (Rodriguez et al., 2005; Lämmermann and Sixt, 2009; µm/h) creeping of adherent animal cells, including fbroblasts Paluch and Raz, 2013). Some crawling cells require dedicated and epithelial cells (Petrie and Yamada, 2015). These cells adhesion molecules to make specifc, high-affnity contacts with move by extending across a surface a sheet-like protrusion their surroundings, whereas other cells rely on weaker, nonspe- called a lamellipodium while also gripping substrate molecules THE JOURNAL OF CELL BIOLOGY CELL OF JOURNAL THE cifc interactions. Crawling cells also use different mechanisms to using integrins, which are often clustered into large focal adhe- advance their leading edge, either assembling polymerized actin sions. Although clinically and physiologically important, this networks to push the plasma membrane forward or detaching the form of adhesion-based crawling is unique to the animal lin- membrane from the underlying cytoskeleton to form a rapidly ex- eage and is largely restricted to molecular highways formed by panding bleb. Furthermore, some cell types have been shown to use the extracellular matrix. contractile forces to generate forward movement (Lämmermann In contrast, many motile cells—including free-living et al., 2008; Bergert et al., 2012; Liu et al., 2015). Different cells amoebae and human immune cells—make 3D actin-flled pseu- can also use different sets of molecules to drive similar modes of dopods and navigate complex environments at speeds exceed- crawling. In an extreme example, nematode sperm have evolved ing 20 µm/min (100–1,000× faster than fbroblasts) without a method of crawling in which polymer assembly advances the forming specifc molecular adhesions (Buenemann et al., 2010; Butler et al., 2010). Although this mode of fast cell crawling Correspondence to Lillian K. Fritz-Laylin: [email protected]; or R. Dyche has been called “ameboid motility,” this term is also used to Mullins: [email protected] describe a range of behaviors, including cell motility that relies L.K. Fritz-Laylin’s present address is Dept. of Biology, University of Massachu- on membrane blebs rather than actin-flled pseudopods (Läm- setts, Amherst, MA 01003. mermann and Sixt, 2009). Abbreviations used: Bd, Batrachochytrium dendrobatidis; fMLP, fMet-Leu-Phe; KD, knockdown; N-WASP, neural WASP; SCAR, suppressor of cAMP recep- tor; TIRF, total internal reflection fluorescence; WASP, Wiskott-Aldrich syndrome © 2017 Fritz-Laylin et al. This article is available under a Creative Commons License (Attribution protein; WAVE, WASP family verprolin-homologous protein. 4.0 International, as described at https ://creativecommons .org /licenses /by /4 .0 /). Supplemental Material can be found at: /content/suppl/2017/05/03/jcb.201701074.DC1.html The Rockefeller University Press $30.00 J. Cell Biol. Vol. 216 No. 6 1673–1688 https://doi.org/10.1083/jcb.201701074 JCB 1673 Published May 4, 2017 To narrow our focus, we use the term “α-motility” specif- neither family is found in all eukaryotic lineages, making them cally to describe cell crawling that is characterized by: (i) highly appealing candidates for genetic markers of α-motility. dynamic 3D pseudopods at the leading edge that are flled with SCAR is generally accepted to play a major role in the branched actin networks assembled by the Arp2/3 complex; formation of protrusions used for cell motility (Miki et al., (ii) fast migration typically on the order of tens of µm/min; and 1998; Steffen et al., 2004; Weiner et al., 2006; Veltman et al., (iii) the absence of specifc, high-affnity adhesions to the extra- 2012). In contrast, the involvement of WASP genes (particu- cellular environment. This independence from specifc molecular larly the two mammalian homologues WASP and N-WASP) in adhesions separates α-motility from the adhesion-based motility cell crawling is less clear. N-WASP is ubiquitously expressed of fbroblasts and epithelial cells. Furthermore, the use of pseu- in mammals and is dispensable for lamellipodia or flopodia dopods discriminates it from the fast bleb-based motility adopted formation by adherent fbroblasts (Lommel et al., 2001; Snap- by fbroblasts in environments that preclude adhesion formation per et al., 2001; Sarmiento et al., 2008), which has led many (Liu et al., 2015; Ruprecht et al., 2015). Some organisms using researchers to discount a role for any WASP protein in protru- α-motility may also use additional methods of generating forward sions or motility (Small and Rottner, 2010; Veltman and Insall, movement, such as contractility, retrograde fow, and/or blebbing 2010). Mammalian WASP, however, is expressed only in blood (Yoshida and Soldati, 2006; Lämmermann et al., 2008; Bergert et cells, where it has been shown to be involved in migration and al., 2012), but in this study, we focus on a single phenotype read- pseudopod formation (Badolato et al., 1998; Burns et al., 2001; ily observable in diverse species, including nonmodel organisms. Jones et al., 2002, 2013; Shi et al., 2009; Ishihara et al., 2012). Organisms with cells capable of α-motility appear Further evidence for a role of WASP in cell migration comes throughout the eukaryotic tree, and we hypothesize that this from the handful of papers studying WASP in nonmammalian form of locomotion refects a single, discrete process that arose cells (Veltman et al., 2012; Zhu et al., 2016; and see Tables S1 early in eukaryotic evolution and has been conserved. If this and S2 for an annotated bibliography of the 29 papers on WASP hypothesis is correct, then elements of this ancient process— and N-WASP relating to cell migration summarized here). The specifc molecules and mechanisms—may be conserved and use of WASP by highly motile cells but not by adherent fbro- Downloaded from still associated with cell crawling in distantly related organisms blasts may therefore refect unique requirements for α-motility. that use α-motility. Such molecular remnants would help to un- To understand the regulation of the actin cytoskeleton ravel the evolutionary history of cell locomotion and might en- during pseudopod formation, we exploit the diversity of organ- able us to predict the existence of specifc modes of motility in isms that use α-motility. By comparing the genomes of many poorly characterized species. Identifying genes associated with eukaryotes, we fnd that organisms with genes encoding both a process such as α-motility is not trivial because the core ma- WASP and SCAR make pseudopods, and organisms that do not chinery driving pseudopod formation (e.g., actin and the Arp2/3 build pseudopods have lost either or both Arp2/3 activators. We complex) is shared with other cellular processes, including validate this molecular signature using a negative test (deplet- on June 21, 2017 some types of endocytosis (Winter et al., 1997). The partici- ing the protein disrupts pseudopod formation in well-studied pation of these proteins in multiple essential processes likely cells) as well as a positive test (a new prediction of α-motility explains their ubiquity in the eukaryotic family tree. Actin, for in a little-studied organism). Differentiating α-motility from example, is found in the genomes of all eukaryotes and, based slow/adhesive cell migration helps clarify the confusion over on phylogenetic analysis, is widely accepted to have been pres- WASP’s importance in cell motility and shifts the major ques- ent in the eukaryotic ancestor (Goodson and Hawse, 2002). The tion from whether WASP or SCAR is required for motility in a Arp2/3 complex is also highly conserved (Beltzner and Pollard, given single cell type to how WASP and SCAR work together to 2004).

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